Rapid dual direct fluorescent antibody assay for the identification of bacillus anthracis

ABSTRACT

In this application is described a method for rapidly and accurately identifying B. anthracis in a sample by simultaneously detecting the presence of cell wall antigen and capsule antigen in the same sample culture grown under capsule inducing conditions. Other uses and advantages of the method of the invention are described herein.

INTRODUCTION

The direct fluorescent antibody assay (DFA) is a procedure that usesantibodies tagged with fluorescent dyes to detect the presence ofspecific antigens on the surface of a cell or microorganism. Recognizedantigens are visible when examined microscopically using a light sourcewhich causes the antibody-bound fluorophore to emit a specificwavelength.

A two-component DFA assay, using fluoroscein-labeled monoclonalantibodies specific to the Bacillus anthracisgalactose-N-acetylglucosamine polysaccharide (Gal-NAG-PS) in the cellwall and poly-D-glutamic acid (CAP) capsule antigen is currentlyavailable for detection of Bacillus anthracis in a sample. Althoughother Bacillus species produce poly-D-glutamic acid capsular material(i.e. certain B. subtilis and B. licheniformis strains), and two B.cereus strains produce the galactose-N-acetylglucosamine polysaccharide(Ezzel et al., 1990, J. Clin. Microbial 28, 223-231), the combination ofboth traits is strongly indicative of B. anthracis.

The current method requires that the test sample is incubated under twodifferent growth conditions: one which greatly reduces capsule formationof B. anthracis (on sheep agar, at 35-37° C., under atmospheric CO2conditions for 16-24 hours), and one allowing capsule formation (20% CO2supplement is frequently used). This is necessary due to the observationthat the anti-Gal-NAG-PS monoclonal antibody is inhibited from reactingwith the cell wall when the capsule is present. Consequently, eachantigen in the DFA assay is detected in a separate vial, each vialcontaining a different subculture of the sample. The necessity fordifferent culture conditions of the sample and separate immunoassayvials for each antigen increases the time and cost required to completethe assay, and allows for misinterpretation of results.

What is needed is a more efficient and less cumbersome method whichallows the detection of both B. anthracis antigens in one vial orculture. Eliminating the need for multiple culture conditions increasesthe rapidity and accuracy of B. anthracis identification. The presentinvention addresses this and other unfulfilled needs in the art.

SUMMARY OF THE INVENTION

In this application is disclosed a novel and rapid detection method forsimultaneously detecting two of the indentifying indicators of Bacillusanthracis, the cell wall polysaccharide Gal-NAG-PS and the capsulepoly-D-glutamic acid.

In order to design an assay for detection of both components, theinventors needed to understand why the Gal-NAG-PS monoclonal did notstain the cell wall in the presence of the capsule. Characterization ofthe B. anthracis capsule using fluorescent dextran beads of varyingmolecular weights revealed that the capsule polypeptides assembled intoa molecular sieve with decreasing density as the distance from the cellwall increased. The inventors determined that dextran beads with stokesradii approximating those of full size immunoglobulin's (IgG and IgM)were unable to penetrate through the capsule layers to reach the cellwall. Conversely, by fluorescently labeling the 5.16 kDa antimicrobialpeptide HBD-3, the inventors determined that this small peptide was ableto penetrate through the capsule layers to bind the cell wall. Theseresults provided one explanation for the inability to detect proteinmolecules binding to, or present on, thE bacterial cell wall usingcomplete IgG or IgM antibodies.

To investigate the possibility that a smaller antibody molecule may ableto penetrate the capsule, the inventors fragmented the anti-Gal-NAG-PSmonoclonal antibody (IGM type, 900 KDa) to produce Fab fragments (50KDa) and tested if the Fab fragment can penetrate the capsule layer, andreact with its antigen in the cell wall. Surprisingly, the Fab fragmentwas able to permeate through the capsule and bind to the cell wallantigen even in the presence of bound anti-CAP antibody.

Therefore, the present invention provides a method for identifying thepresence of Bacillus anthracis in a sample by simultaneously identifyingthe presence of capsule and cell wall in one sample. The methodencompasses the addition of detectably labeled antibody Fab fragmentspecific for a cell wall antigen in tandem with differentiallydetectably labeled anti-capsule antibody, in a microbial culture from abiological, environmental, or forensic sample, and detecting thepresence of complexes formed such that presence of label specific forthe anti-cell wall Fab indicates cell wall, and presence of labelspecific for anti-capsule antibody indicates capsule, and presence ofboth Labels strongly indicates the presence of B. anthracis in thesample. In a specific embodiment, the cell wall Fab antibody isanti-Gal-NAG-PS. In another specific embodiment, the capsule antibody isanti-poly-D-glutamic acid.

The present invention provides several advantages. In the presentinvention, a single incubation under capsule inducing conditions can beused for detecting both the B. anthracis cell wall antigen and capsuleantigen, eliminating the need for additional subcultures at varyingconditions. Staining the same bacillus with antibodies conjugated withdifferent labels, e.g. contrasting fluorophores, confers accuracy andminimizes misinterpretation of results. In addition, the presentinvention allows the identification of some atypical isolates of B.anthracis which produce capsule under atmospheric CO2 conditions, aswell as aberrant Bacillus such as the pathogenic B. cereus that do notproduce the anthracis poly-D-glutamic acid capsule, but do contain theanthracis-like cell wall polysaccharide. Previously, use of theGal-NAG-PS IgM would not detect the cell wall, however, theanti-Gal-NAG-PS Fab antibody of the present invention would overcomethis limitation.

Another major advantage of the herein described tandem DFA protocol isthat it encourages the immediate analysis of any incoming culture orblood sample, without further culturing. Much information can begathered with a first look at the sample: if it contains B. anthracis,the cell wall will be detected and the sample will be pCHO positive.pCHO is B. anthracis plasmid encoding the synthesis of cell wallN-acetyl-D-glucosamine, Gal-NAG polysaccharide. If it contains capsule,the sample will be pX02 positive, and the capsule laden cells will bedetected. pX02 is a B. anthracis plasmid encoding the synthesis ofpoly-D-glutamyl capsule. This is a quantum leap for early identificationand/or verification of B. anthracis in a sample. Further confirmationwould follow about 3 hours later post incubation under capsule producingconditions.

Therefore, it is one object of the present invention to provide a methodfor identifying the presence of Bacillus anthracis in a sample bysimultaneously identifying the presence of capsule and cell wall, byaddition of detectably labeled anti-Gal-NAG-PS Fab fragment in tandemwith differentially detectably labeled anti-CAP antibody, to thebiological sample, microbial culture or environmental sample, anddetecting the presence of complexes formed such that presence ofanti-Gal-NAG-PS label indicates cell wall and presence of anti-capsule(anti-CAP) label indicates capsule and presence of both labels stronglyindicates the presence of B. anthracis in the sample.

It is another object of the invention to provide novel test kits fordetection of B. anthracis comprising antibodies according to the presentinvention. The antibodies are directly or indirectly attached to asuitable reporter molecule, e.g., an enzyme, a radionuclide, or afluorophore. The test kit includes a container holding one or moreantibodies according to the present invention and instructions for usingthe antibodies for the purpose of detecting B. anthracis in a sample bydetecting the formation of the immunological complex(es) such thatpresence or absence of the immunological complex(es) correlates withpresence or absence of B. anthracis.

It is still another object of the present invention to provide a methodfor targeting therapeutic compounds to B. anthracis. The anti-Gal-NAG-PSand anti-CAP antibodies of the present invention can be complexed totherapeutic compounds, e.g. one or more bactericidal compound or toxin,such that the compounds are delivered to the cell wall or capsule uponcontact with B. anthracis. Some examples of compounds include2-epimerase enzymes, e.g. 2-epimerase (Schuch et al., 2013, In Aziz,Ramy K. PLoS ONE 8(4):e60754; Vellosos et al, 2008, EMBO Rep 9: 199-205)and phage endolysines e.g. PlyG, or active portions thereof (Ganguly etal., 2013 Glycobiology 23, 820-32).

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIG. 1. Schematic for the Original vs Tandem DFA assay. The originalmethod required an overnight SBA culture under atmospheric CO2. Fromthat culture the pCHO DFA could be directly performed. However, it wasthen necessary to create a 2nd culture from this overnight in a capsuleinducing broth medium for about 4 hours. The tandem assay of the presentinvention requires only one growth condition in one tube, and one slidefor simultaneous observation of capsule and cell wall staining.

FIG. 2. Representative results using the tandem DFA assay of the presentinvention showing B. anthracis detected in sample under capsule inducingconditions using bright field microscopy and fluorescence detection(cell wall and capsule). Set A: represents potential staining pattern ofwild type virulent strains, i.e. B. anthracis Sterne. Set B: representspotential staining pattern of pxo1−, pX02+ strains, i.e. Pasteurcontrols.

DETAILED DESCRIPTION

In the description that follows, a number of terms are extensivelyutilized. In order to provide a clearer and consistent understanding ofthe specification and claims, including the scope to be given suchterms, the following definitions are provided.

As used herein, the term “antigen” is a molecule capable of being boundby an antibody. Thus, antigenic determinants or epitopes are those partsof an antigen that are recognized by antibodies. An antigenicdeterminant need not be a contiguous sequence or segment of protein andmay include various sequences that are not immediately adjacent to oneanother.

As used herein the terms “specific to” or “specific for” a targetsequence, in relation to an antibody, relate to an antibody or antibodyfragment that binds an antigen, under conditions used in givencircumstances (e.g., temperature, salt concentration, etc.), but doesnot significantly bind to other antigens or polypeptides that are nottarget antigens.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

It also is specifically understood that any numerical value recitedherein includes all values from the lower value to the upper value,i.e., all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application. For example, if a range is statedas 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%,or 1% to 3%, etc., are expressly enumerated in this specification.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., 1995, ProteinEng. 8(10): 1057-1062); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fe” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen binding site. This region consists of adimer of one heavy-chain and one light-chain variable domain in tight,non-covalent association. It is in this configuration that the threeCDRs of each variable domain interact to define an antigen-binding siteon the surface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue (s) of the constantdomains bear a free thiol group. F(ab′) 2 antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, andIgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the sFvto form the desired structure for antigen binding. For a review of sFv,see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or polysaccharide or an epitope on a particularpolypeptide or polysaccharide is one that binds to that particularpolypeptide or polysaccharide or epitope on a particular polypeptide orpolysaccharide without substantially binding to any other polypeptide orpolypeptide epitope.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The word “subject” includes human, animal, avian, e.g., horse, donkey,pig, mouse, hamster, monkey, chicken, sheep, cattle, goat, buffalo, andany other subject suspected of being infected with B. anthracis.

The language “biological sample” is intended to include biologicalmaterial, e.g. cells, blood, tissues, biological fluid, or a solutionfor administering to a subject, such as a vaccine, or immunoglobulin. By“environmental sample” is meant a sample such as soil and water. Foodsamples include canned goods, meats, milk, and other suspectedcontaminated food. Forensic sample includes any sample from a suspectedterrorist attack suspected of having anthrax, including paper, powder,envelope, container, hair, fibers, and others.

Polyclonal Antibodies. The antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan. Polyclonal antibodies canbe raised in a mammal, for example, by one or more injections of animmunizing agent and, if desired, an adjuvant. Typically, the immunizingagent and/or adjuvant will be injected in the mammal by multiplesubcutaneous or intraperitoneal injections. The immunizing agent mayinclude the polypeptide or a fusion protein thereof. It may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude but are not limited to keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, and soybean trypsin inhibitor. Examples ofadjuvants which may be employed include Freund's complete adjuvant andMPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

Monoclonal Antibodies. The antibodies may, alternatively, comprisemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the polypeptide orpolysaccharide or a fusion protein thereof. Generally, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, 1984, J. Immunol., 133:3001; Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thedesired antigen. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, 1980, Anal. Biochem. 107:220.

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fe region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe B. anthracis antigen, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, 1983, Nature, 305:537-539). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., 1991, EMBO J., 10:3655-3659.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., 1986, Methods in Enzymology, 121:210.

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain (s) are created on the interface of the second antibody moleculeby replacing large amino acid side chains with smaller ones (e.g.alanine or threonine). This provides a mechanism for increasing theyield of the heterodimer over other unwanted end-products such ashomodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)z bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., 1985,Science 229:81 describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., 1992. J. Exp.Med. 175:217-225 describe the production of a fully humanized bispecificantibody F(ab′) 2 molecule. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., 1992, J. Immunol. 148 (5):1547-1553.The leucine zipper peptides from the Fas and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., 1993, Proc. Natl.Acad. Sci. USA 90:6444-6448 has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the Vh and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,1994, J. Immunol. 152:5368.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., 1991, J. Immunol.147:60.

Exemplary bispecific antibodies may bind to two different epitopes on agiven B. anthracis antigen herein. Alternatively, an anti-B. anthracisantigen arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fe receptors for IgG (FcyR), such as FcyRI (CD64),FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defensemechanisms to the bacilli. Bispecific antibodies may also be used tolocalize cytotoxic agents to B. anthracis bacilli, or other bacilli withCAP or Gal-NAG-PS. For example, these antibodies possess a cell wallbinding arm and an arm which binds a cytotoxic agent or a bactericidalagent, for example, epimerox or PlyG.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a bactericidal agent such as an antibiotic or an enzymewhich is able to digest part or all of the cell wall, for example,Epimerox or PlyG.

Conjugates of the antibody and bactericidal agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and his-active fluorine compounds (suchas1,5-difluoro-2,4-dinitrobenzene).

The present invention pertains to a method for detecting B. anthracis ina sample suspected of containing B. anthracis. The method includesculturing the sample under capsule inducing conditions. Capsule-inducingconditions are well known to people in the art, see Examples below.After culturing, the sample is contacted simultaneously with twoantibodies, one Fab antibody specific for cell wall and one antibodyspecific for the capsule, allowing the antibody to bind to its antigento form an immunological complex, detecting the formation of theimmunological complex of each antibody, and correlating the presence ofeach immunological complex with the presence of B. anthracis in thesample. The sample can be biological, environmental, forensic, or a foodsample.

Two monoclonal antibodies are described herein. The development andcharacterization of the monoclonal antibody to B. anthracis cell wallgalactose-N-acetylglucosamine polysaccharide, also known as EAII-6G6,was described by Ezzell et al., 1990, Journal of Clinical, Microbiology,volume 28, pages 223-231. Briefly, guanidine extracts of crude Bacillusanthracis cell wall were used to vaccinate BALB/c mice and to developmonoclonal antibody to vegetative cell surface antigens. Two Hybridomaswere sleeted which produced immunoglobulin M immunoglobulin's, directedto an epitope associated with the galactose-N-acetyl-D-glucosaminepolysaccharide. Both demonstrated specificity in their binding topurified B. anthracis cell wall, o-stearoyl-polysaccharide conjugates,and intact nonencapsulated vegetative cells. Electron microscopy showedthat both monoclonal antibodies interacted with the cell wall ofvegetative cells as well as with the cortex of spores. Neithermonoclonal reacted with encapsulated vegetative cells nor with intactspores. After conjugation to fluorescein isothiocyanate, the monoclonalsstained intensely all B. anthracis strains tested, some strains ofBacillus cereus, but non of the 20 other Bacillus spp.

The original form, whole IgM, of the antibody has been used for about 20years by the Department of Defense. In the present invention, a Fabfragment of the anti-Gal-NAG-PS cell wall IgM was used as described inthe Examples below. Methods for production of Fab fragments from wholeantibody molecules is known in the art. The anti-Gal-NAG-PS cell wallIgM was digested into Fab fragments and subsequently labeled.

Methods for making monoclonal antibodies to the poly-D-glutamic acid(PDGA)polypeptide are known in the art. See for example Kozel et al.,2007, Infect. Immun. 75, 152-163 or Kozel et al., 2004, PNAS 101,5042-5047.

The monoclonal antibodies used in the herein described invention FDF-1B9(De, B. K. et al., Emerg. Infect. Dis. 2002, 8:1060-65), can potentiallybe replaced with another PDGA monoclonal known to bind capsule. Thesubstituted monoclonal antibody should be able to bind the PDGA and notinterfere with binding of the cell wall monoclonal antibody.

Similarly, other monoclonal antibodies which specifically bind the cellwall galactos-N-acetylglucosamine polysaccaride can be used.Characteristics of such a monoclonal and methods of preparing same arefound in Ezzell, et al., 1990, J. Clin. Microbial. 28, 223-231.

The antibodies used in the diagnostic assays described herein can belabeled with a detectable moiety. The detectable moiety should becapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, auramine, TEXAS RED (sulforhodomine 101 acidchloride), AMCA blue, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., 1962,Nature, 144:945; David et al., 1974, Biochemistry, 13: 1014; Pain etal., 1981, J. Immunol. Meth., 40:219; and Nygren, 1982, J. Histochem.and Cytochem., 30:407. Labels include, but are not limited to moietiesthat are directly or indirectly detectable such as radioactive elements,enzymes, fluorescent molecules or chemicals, and others. In oneembodiment, the Fab fragment is labeled with a fluorophore,specifically, Dylight 594, as described in the Examples. Other labelscan be used as is known to people with skill in the art.

The present invention also provides kits which are useful for carryingout the present invention. The present kits comprise a first containermeans containing the above-described antibodies. The kit also comprisesother container means containing solutions necessary or convenient forcarrying out the invention. The container means can be made of glass,plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. Thekit may also contain written information, such as procedures forcarrying out the present invention or analytical information, such asthe amount of reagent contained in the first container means. Thecontainer means may be in another container means, e.g. a box or a bag,along with the written information.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors and thought to function well inthe practice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

All documents cited herein are hereby incorporated in their entirety byreference thereto.

Example 1

Molecular Architecture of B. anthracis Capsule

Methods

Encapsulated B. anthracis bacilli (Ames strain), or the unencapsulatedmes CapA derivative were incubated with FITC labeled dextrans (green)(FDx, Molecular Probes, Life Technologies, Frederick, Md.) for 30minutes at room temperature. Bacterial DNA was then stained with DAPI(blue). Images were acquired using a TE2000 microscope equipped withphase contrast and fluorescence filters. Images were overlaid and thepixel intensity of FDx fluorescence was determined across the center ofthe cells using In-Vivo Imaging software (Media Cybernetics, Rockville,Md.). Fab antibodies were prepared from anti-capsule monoclonal antibodyFDF-1B9 (USAMRIID) using a commercially available kit. Products werecharacterized by size exclusion chromatography using an FPLC system anda Superose 6 SEC column (Amersham Pharmacia, Piscataway, N.J.), in PBSwith monitoring at 280 nm. Anti-capsule IgM was labeled with FITC(greenfluorescence), and anti-capsule Fab was labeled with ALEXAFLUOR(carboxamido-(6-azidohexanyl, triethylammonium salt) (hereafter referredto as AF594) (red fluorescence).

Results

The results showed that 3 kDa and 500 kDa FDx were not impeded fromreaching the poly-D-gamma-glutamic acid of an unencapsulated strain(CapA, figure not shown). In contrast, when using the encapsulated Amesstrain, the line intensity profiles revealed that the peaks of FDxfluorescence were further from the cell wall for the 40 kDa and 500 kDaFDx than for the 3 kDa FDx. These results suggested that FDx ofincreasing molecular size were blocked from reaching the cell walls ofencapsulated bacilli by PGGA.

In previous studies, we determined that intact antibodies, (IgG andIgM), were not able to completely penetrate the capsule to recognizeantigens on the cell wall. Considering the results obtained of the FDxexperiment, we predicted that IgG antibody having a Stokes radius of 5.5nm, which is larger than the excluded 40 kDa FDx, would have limitedaccess to the bacillus cell wall. In contrast, an antibody Fab fragmentwith a Stokes radius of 3.2 nm closely approximates that of a 10 kDa FDx(2.8 nm), and should therefore have better access to the interior ofcell wall than an intact IgG or IgM antibody.

We generated Fab fragments from anti-capsule mAb FDF-1B9 (USAMRIID,Frederick, Md.). A limited digestion resulted in 2 major peaks asdetermined by size exclusion chromatography (figure not shown). Peak 1corresponded to the predicted size of IgM, and peak 2 to the predictedsize of Fab fragments. In order to determine if anti-capsule IgM or Fabfragment antibodies bind to distinct regions of the capsule, weincubated encapsulated bacilli with intact FITC labeled anti-capsule mAb(IgM), and measured the pixel intensities of a line drawn through thecenter of the bacillus (figure not shown). We next incubated a separatealiquot of the same bacilli with AF594 labeled Fab and performed thesame analysis (figure not shown). Results of the binding of IgM comparedto Fab showed marked differences. The fluorescence peaks representingthe maximum pixel intensities of IgM FITC binding were significantlyfarther away from the bacilli than the peaks for Fab binding (figure notshown).

As predicted, these results suggested that the anti-capsule IgM wasbinding poly-D-gamma-glutamic acid (PGGA) exterior to the anti-capsuleFab. In order to verify that this was the case, we incubatedencapsulated bacilli with AF594 labeled Fab for 15 minutes first, washedaway unbound antibody, then incubated the bacilli with FITC labeled IgMfor 15 minutes (figure not shown). Images were captured and afluorescence intensity profile was generated by drawing a line acrossthe bacillus and graphing the pixel intensities. The analysis revealedthat the FITC labeled IgM was deposited on the surface of the PGGAcapsule, and farther away from the cell wall than the binding of AF594Fab. In addition, incubation with FITC IgM after incubation with AF594Fab changed the staining pattern for AF594 Fab from a sharp rim pattern(figure not shown) to a diffuse pattern with no clear zone between theAF594 Fab signal and the cell wall. These results suggested that whileanti-capsule IgM binds to the exterior of the capsule, it also changesthe structure of the interior layers of the capsule.

Conclusions

The B. anthracis poly-D-glutamic acid capsule functions as a molecularsieve. The capsule has a variable porosity that increases with distancefrom the cell wall. A high density of the polypeptide at the capsuleinterior prevents penetration of large macromolecules to sites near thecell wall. In contrast, the capsular edge consists of capsularpolypeptide in a lower density that exhibits increased permeability tomacromolecules. Macromolecules having Stokes radii of greater than, orequal to 4.6 nm (i.e. IgG and IgM) are impeded from reaching the B.anthracis bacillus cell wall by the PGGA capsule. Fragmentation ofantibodies into Fab fragments facilitates the penetration of antibodiesthrough the PGGA capsule to reach the bacillus cell wall.

Example 2 Tandem DFA Assay

Materials and Equipment

-   -   5% sheep blood agar (SBA) plates    -   PBS (sterile)    -   Fluoromount G (or equivalent quench/mountant)    -   Anti-B. anthracis pCHO FAb Dylight 594 DFA reagent    -   Anti-B. anthracis capsule IgM FITC DFA reagent    -   Incubator (equipped for 20% CO2 supplementation), 35-37° C.    -   B. anthracis Pasteur (positive control: spores or vegetative        cells)    -   B. cereus NCTC 2599 or equivalent (negative control: spores or        vegetative cells)    -   Test sample (s)    -   Epifluorescent microscope equipped with filters (EF-4 TEXAS RED        (sulforhodomine 101 acid chloride) HyQ or equivalent for Dylight        594; B-2 E/C or equivalent for FITC)    -   Bleach rite (or equivalent NA Hypochlorite disinfectant)

Procedure “Quick Look” Assay

Note: It is highly recommended to perform a STAT tandem DFA on theincoming sample when presented in a vegetative cell form. Earlydetection of target antigens will expedite results. A positive pCHOresult is indicative of Bacillus anthracis; it is very possible thatsufficient capsule is present for a positive DFA result. Continue theincubation as per the procedure, but initial results should be reported.

Criteria to consider for determining potential ‘quick look’ DFA:

-   -   1) Organisms in sample must be in vegetative cell form    -   2) Must be less than 24 hours old    -   3) Must have sufficient vegetative cells available        -   a. Ideally presented on 5% SBA perform quick morphologic            examination            -   i. Non-hemolytic            -   ii. Ground glass in appearance            -   iii. Sufficient vegetative cells can be tested from                individual colony(ies).        -   b. If presented in broth form            -   i. Microscopic examination of wet mount revealing                bacillus possibly single to multiple cells/chain            -   ii. Positive Gram stain            -   iii. Purity of wet mount examination of sample (are                other bacteria present)            -   iv. Depending on number of cells per field of view and                other non-bacillus anthracis cells present may need to                concentrate cells so low frequency of B. anthracis cells                are not ‘missed’. If cell concentration is too low,                cells may be pelleted via centrifugation and resuspended                ins μl of residual liquid at end of assay.

On Slant with No Isolated Colonies:

Remove 1 μl loopful of culture material, transfer to 100 μl of sterilePBS and resuspend well. (Resuspended Material Here could be used for“quick look” then proceed to remainder of DFA procedure).

On 5% SBA or Other Medium Type with Isolated Colonies:

Remove 1 μl loopful of isolated colony culture material (or entirecolony if insufficient material to fill loop) and transfer to 100 μl ofsterile PBS; this constitutes the inoculum. Resuspend the inoculum well.(Resuspended Material Here could be used for “quick look” then proceedto remainder of DFA procedure).

Tandem DFA for “Quick Look”—

-   -   Transfer 10 μl of the inoculum from above to a 250-500 μl        eppendorf tube. Repeat for each inoculum and controls (NOTE:        cannot perform STAT DFA on spores, only vegetative cells).        Perform DFA assay as noted below.    -   In separate tubes, dilute 5 μl of each test and control cells        with 5 μl of PBS. To each tube, add 2.5 μl of the pCHO FAb        Dylight 594 DFA reagent and mix with pipet tip. Allow to sit for        approximately 5 minutes at room temperature and then to each        tube, add 2.5 μl of the capsule IgM FITC DFA reagent and repeat        mixing with pipet tip. This constitutes the DFA reaction tube.        Continue to incubate at room temperature (or at 35-37° C.) for        an additional 5-10 minutes.    -   Post-incubation, add 20 μl of PBS to the DFA reaction tube and        mix with pipet tip to ‘wash’.    -   To clean tube, place approximately 2 μl of Fluorogel quench,        then immediately transfer approximately 1 μl of DFA reaction        tube and mix well with pipet tip. Note: the quench to reaction        tube ratio should be 2:1; adjust volumes accordingly. Transfer        approximately 2 μl of the quench/DFA reaction material to        microscope slide and add cover slip.    -   Examine cells using epifluorescent microscopy equipped with        appropriate filters (EF-4 TEXAS RED (sulforhodomine 101 acid        chloride) HyQ or equivalent for viewing Dylight 594 conjugated        anti-pCHO FAb; B-2E/C or equivalent for FITC conjugated        anti-capsule IgM)

Regular DFA Assay Incoming Sample (s):

As Spores:

Unknown purity of sample dictates that material be plated onto 5% SBA,streaked for isolation and cultured for 18-24 hrs @ 35-37° C. undersupplemented 20% CO2 prior to DFA procedure. At this time, B. anthracisPasteur and B. cereus NCTC 2599 controls should also be plated (fromspore form if available).

As Environmental Sample in Liquid Form:

Plate 50 μl of liquid onto 5% SBA and streak for isolation. Incubate at35-37° C. for 18-24 hrs.

From Slant with No Isolated Colonies:

Remove 1 μl loopful of culture material, and streak for isolation on 5%SBA. Incubate at 35-37° C. for 18-24 hrs.

On 5% SBA or Other Medium Type with Isolated Colonies:

Remove 1 μl loopful of isolated colony culture material (or entirecolony if insufficient material to fill loop) and streak for isolationon fresh 5% SBA. Repeat on several suspect colonies. Incubate at 35-37°C. for 18-24 hrs.

Controls: B. anthracis Pasteur and B. cereus NCTC 2599:

Plate 1 μl of stock spore suspension (B. anthracis Pasteur and B. cereusNCTC 2599 stocks 10⁷ to 10⁹ spore/ml) onto SBA plate and streak forisolation. Incubate all cultures at 35-37° C. for 18-24 hrs.

Tandem DFA

Inspect colony formations on 18-24 hr 5% SBA cultures. Suspect coloniesshould mimic the general characteristics of B. anthracis such as, butnot to be restricted to, colonies exhibiting non-hemolytic, raised, 3-6mm diameter, round to slight oblong shape, grayish white to tannishcolor, ground glass appearance with possible Medusa head formations.

From 18-24 hr 5% SBA cultures, remove 1 μl loopful of isolated suspectcolony culture material (or entire colony if insufficient material isavailable to fill loop) and transfer to 100 μl of sterile PBS; thisconstitutes the inoculum). Resuspend the inoculum via gentle vortexingor mixing with uptake and release of suspension with pipet.

Transfer 50 μl of the inoculum to surface of 5% SBA plate. Using end ofinoculation loop (or disposable spreader), spread liquid in lawn fashionover ⅓ to ½ of surface area until absorbed into agar. Repeat steps withseveral isolated colonies on separate 5% SBA plates. Repeat steps with 2colony representatives from each control plate. Incubate all plates at35-37° C. under 20% supplemented CO2 for 2-2.5 hours (2.5 hrs maximum).

Post 2-2.5 hr incubation of cultures, apply 100 μl of sterile PBS ontosurface of agar where ‘frosty’ appearance of growth is visible. Usinginoculating loop, scrape off growth within a quarter size area, thentilt plate to retrieve the vegetative cell rich suspension; thisconstitutes the test cells. Transfer suspension to clean labeledeppendorf tube; hold on ice if cannot perform DFA immediately. Repeatfor each sample and control culture.

In separate tubes, dilute 5 μl of each test and control cells with 5 μlof PBS. To each tube, add 2.5 μl of neat anti-B. anthracis pCHO FAbDylight 594 DFA reagent and mix with pipet tip. Allow to sit forapproximately 5 minutes at room temperature and then to each tube, add2.5 μl of the anti-B. anthracis capsule IgM FITC DFA reagent and repeatmixing with pipet tip. This constitutes the DFA reaction tube. Continueto incubate at room temperature for an additional 5-10 minutes.Alternatively, tubes can also be incubated at 35-37° C. for 5-10minutes.

Post-incubation, add 20 μl of PBS to the DFA reaction tube and mix withpipet tip to wash.

To clean tube, place approximately 2 μl of Fluoromount G quench, thenimmediately transfer approximately 1 μl of DFA reaction tube and mixwell with pipet tip. Note: the quench to reaction tube ratio should be2:1; adjust volumes accordingly. Transfer approximately 2 μl of thequench/DFA reaction material to microscope slide and add cover slip.

Repeat steps for each test and control sample one at a time as theFluoromount G will solidify rather quickly.

Examine cells using epifluorescent microscopy equipped with appropriatefilters (EF-4 TEXAS RED (sulforhodomine 101 acid chloride) HyQ orequivalent for viewing Dylight 594 conjugated anti-pCHO FAb; B-2E/C orequivalent for FITC conjugated anti-capsule IgM).

Individual cell(s) are examined for both epifluorescent signalssimultaneously via switching of filters. Cell (s) positive for capsulewill appear brilliant green; positively stained B. anthracis pCHO ofvegetative cells will emit bright red either along periphery ofvegetative cells or just at septa; negative pCHO staining will result in‘shadow’ effect. A positive pX02+ B. anthracis should stain for bothantigens whereas a pxo2− strain (i.e. B. anthracis Sterne) will onlystain for the pCHO (FIG. 2).

Confirm that the stained cells are intact by examination via brightfield. Vegetative cells that appear ‘empty’ indicate cell death and cellwall integrity compromised thus allowing antibody to possibly enter andbecome ‘trapped’. Vegetative cells should appear opaque under brightfield examination.

Repeat the DFA assay up to 3 hours if the capsule DFA results arequestionable. Failure of capsule production at 3 hours for the B.anthracis Pasteur control indicates a failure within the assay and theentire assay should be repeated with freshly prepared cultures. Althougha poor capsule producer, presence of capsule on the Pasteur straineither in punctuate or entire form along with the staining of thesecondary cell wall pCHO indicates successful culturing and staining viathe tandem antibody(s); negative results exhibited by sample should berendered accurate.

NOTE: Incubation of cultures over 3 hours may introduce extraneousproteins that could interfere with the pCHO antibody binding.Historically, the whole anti-pCHO IgM molecule was used for staining thevegetative cells of anthracis. This form also did not stain 100% ofvegetative cells within a given sample. The question of why was notanswered until the development of the tandem DFA assay. Optimizing theassay revealed issues regarding the deposition of EA1 and/or SAPproteins during late lag/early log phase which masked thegalactose-N-acetyl-glucosamine (pCHO) motif presented on the cell wallof anthracis. In order to utilize the pCHO FAb as intended in tandemwith the capsule MAb, it was necessary to optimize not only the time andgrowth conditions for capsule production, but also the pre-EA1/SAPdeposition. This nwindow” has fallen into a 2-3 hour range.

In addition, the negative and positive controls for the tandem DFA assayhave to be in the same form as the test sample, i.e. the vegetativecells form. This is because, for example, if spores were used for thepositive control vs. vegetative cells as the test sample, the productionof capsule would perhaps be skewed towards the spores; this would not bean absolute comparison for the vegetative cell form sample. Therefore,it is best to confirm that the culture conditions are correct for thepositive control to produce capsule; a poor capsule producer is usedhere in order to have confidence that if the control is able to producecapsule, then the conditions were optimized for the test sample to alsoproduce capsule. The positive control should not be presented in sporeform for it will easily produce capsule and the likelihood of thesubmitted sample being in spore form is low.

What is claimed is:
 1. A kit for determining the presence of B.anthracis in a sample by simultaneously detecting the presence of cellwall antigen and capsule antigen in the same culture grown under capsuleinducing conditions, comprising: antibody one which specifically bindsto a capsule antigen, Fab antibody two which specifically binds to acell wall antigen, wherein antibody one and Fab antibody two arecontrastingly labeled, and means for detecting immune complexes formedbetween antibody one and its capsule antigen, and Fab antibody two andits cell wall antigen.
 2. The kit of claim 1, wherein antibody 1 is mABFDF-1B9.
 3. The kit of claim 1 wherein Fab antibody two is mAbEAII-6G6Fab.
 4. The kit of claim 2, wherein antibody two is mAbEAII-6G6Fab.